scholarly journals Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages

2016 ◽  
Vol 55 (3) ◽  
pp. 865-876 ◽  
Author(s):  
M. D. Cairns ◽  
M. D. Preston ◽  
C. L. Hall ◽  
D. N. Gerding ◽  
P. M. Hawkey ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Antibiotic Resistance Genes ◽  
Binary Toxin ◽  
Human Origins ◽  
Comparative Genome Analysis ◽  
Toxin B ◽  
Content Type ◽  
Global Spread ◽  
Animal Isolates ◽  
Evolutionary Lineages

ABSTRACT The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

scholarly journals Infection with Toxin A-Negative, Toxin B-Negative, Binary Toxin-Positive Clostridium difficile in a Young Patient with Ulcerative Colitis

2015 ◽  
Vol 53 (11) ◽  
pp. 3702-3704 ◽  
Author(s):  
Grace O. Androga ◽  
Julie Hart ◽  
Niki F. Foster ◽  
Adrian Charles ◽  
David Forbes ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Clostridium Difficile ◽  
Young Patient ◽  
Diagnostic Methods ◽  
Binary Toxin ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
Severe Diarrhea ◽  
Clinically Significant

Large clostridial toxin-negative, binary toxin-positive (A−B−CDT+) strains ofClostridium difficileare almost never associated with clinically significantC. difficileinfection (CDI), possibly because such strains are not detected by most diagnostic methods. We report the isolation of an A−B−CDT+ribotype 033 (RT033) strain ofC. difficilefrom a young patient with ulcerative colitis and severe diarrhea.

scholarly journals Toxigenic Clostridium difficile PCR Ribotypes from Wastewater Treatment Plants in Southern Switzerland

2012 ◽  
Vol 78 (18) ◽  
pp. 6643-6646 ◽  
Author(s):  
Vincenza Romano ◽  
Vincenzo Pasquale ◽  
Karel Krovacek ◽  
Federica Mauri ◽  
Antonella Demarta ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Clostridium Difficile ◽  
Wastewater Treatment Plants ◽  
Treatment Plant ◽  
Treated Wastewater ◽  
Binary Toxin ◽  
Toxin B ◽  
Content Type ◽  
Human Infections ◽  
Reuse Of Treated Wastewater

ABSTRACTThe occurrence ofClostridium difficilein nine wastewater treatment plants in the Ticino Canton (southern Switzerland) was investigated. The samples were collected from raw sewage influents and from treated effluents. Forty-seven out of 55 characterizedC. difficilestrains belonged to 13 different reference PCR ribotypes (009, 010, 014, 015, 039, 052, 053, 066, 070, 078, 101, 106, and 117), whereas 8 strains did not match any of those available in our libraries. The most frequently isolated ribotype (40%) was 078, isolated from six wastewater treatment plants, whereas ribotype 066, a toxigenic emerging ribotype isolated from patients admitted to hospitals in Europe and Switzerland, was isolated from the outgoing effluent of one plant. The majority of the isolates (85%) were toxigenic. Forty-nine percent of them produced toxin A, toxin B, and the binary toxin (toxigenic profile A+B+CDT+), whereas 51% showed the profile A+B+CDT−. Interestingly, eight ribotypes (010, 014, 015, 039, 066, 078, 101, and 106) were among the riboprofiles isolated from symptomatic patients admitted to the hospitals of the Ticino Canton in 2010. Despite the limitation of sampling, this study highlights that toxigenic ribotypes ofC. difficileinvolved in human infections may occur in both incoming and outgoing biological wastewater treatment plants. Such a finding raises concern about the possible contamination of water bodies that receive wastewater treatment plant effluents and about the safe reuse of treated wastewater.

scholarly journals Characterization of Temperate Phages Infecting Clostridium difficile Isolates of Human and Animal Origins

2014 ◽  
Vol 80 (8) ◽  
pp. 2555-2563 ◽  
Author(s):  
Ognjen Sekulovic ◽  
Julian R. Garneau ◽  
Audrey Néron ◽  
Louis-Charles Fortier
Keyword(s):  
Antibiotic Resistance ◽  
Clostridium Difficile ◽  
Uv Light ◽  
Antibiotic Resistance Genes ◽  
Soft Agar ◽  
Human Origins ◽  
Content Type ◽  
Double Stranded Dna ◽  
Animal Strains

ABSTRACTClostridium difficileis a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs ofC. difficilethat could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. InC. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and humanC. difficileisolates. Prophages were induced by UV light from 58C. difficileisolates of animal and human origins. Using soft agar overlays with 27 differentC. difficiletest strains, we isolated and further propagated nine temperate phages: two from horse isolates (ϕCD481-1 and ϕCD481-2), three from dog isolates (ϕCD505, ϕCD506, and ϕCD508), and four from human isolates (ϕCD24-2, ϕCD111, ϕCD146, and ϕCD526). Two phages are members of theSiphoviridaefamily (ϕCD111 and ϕCD146), while the others areMyoviridaephages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from humanC. difficileisolates, especially the members of theSiphoviridaefamily, had a broader host range than phages from animalC. difficileisolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported inC. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and humanC. difficileisolates.

scholarly journals A New Type of Toxin A-Negative, Toxin B-Positive Clostridium difficile Strain Lacking a CompletetcdAGene

2014 ◽  
Vol 53 (2) ◽  
pp. 692-695 ◽  
Author(s):  
Sandra Janezic ◽  
Mercedes Marín ◽  
Adoración Martín ◽  
Maja Rupnik
Keyword(s):  
Clostridium Difficile ◽  
Virulence Factors ◽  
Diagnostic Tests ◽  
Integration Site ◽  
Molecular Diagnostic ◽  
Binary Toxin ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
New Type

Toxins A and B are the main virulence factors ofClostridium difficileand are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficiletransferase)-negative (A−B+CDT−) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) withouttcdAand with atypical organization of the PaLoc integration site.

scholarly journals Characterizations of Clinical Isolates of Clostridium difficile by Toxin Genotypes and by Susceptibility to 12 Antimicrobial Agents, Including Fidaxomicin (OPT-80) and Rifaximin: a Multicenter Study in Taiwan

2012 ◽  
Vol 56 (7) ◽  
pp. 3943-3949 ◽  
Author(s):  
Chun-Hsing Liao ◽  
Wen-Chien Ko ◽  
Jang-Jih Lu ◽  
Po-Ren Hsueh
Keyword(s):  
Clostridium Difficile ◽  
Care Setting ◽  
Antimicrobial Agents ◽  
Teaching Hospitals ◽  
Binary Toxin ◽  
Content Type ◽  
Vancomycin Mics ◽  
Major Teaching ◽  
Southern Taiwan

ABSTRACTA total of 403 nonduplicate isolates ofClostridium difficilewere collected at three major teaching hospitals representing northern, central, and southern Taiwan from January 2005 to December 2010. Of these 403 isolates, 170 (42.2%) were presumed to be nontoxigenic due to the absence of genes for toxins A or B or binary toxin. The remaining 233 (57.8%) isolates carried toxin A and B genes, and 39 (16.7%) of these also had binary toxin genes. The MIC90of all isolates for fidaxomicin and rifaximin was 0.5 μg/ml (range, ≤0.015 to 0.5 μg/ml) and >128 μg/ml (range, ≤0.015 to >128 μg/ml), respectively. All isolates were susceptible to metronidazole (MIC90of 0.5 μg/ml; range, ≤0.03 to 4 μg/ml). Two isolates had reduced susceptibility to vancomycin (MICs, 4 μg/ml). Only 13.6% of isolates were susceptible to clindamycin (MIC of ≤2 μg/ml). Nonsusceptibility to moxifloxacin (n= 81, 20.1%) was accompanied by single or multiple mutations ingyrAandgyrBgenes in all but eight moxifloxacin-nonsusceptible isolates. Two previously unreportedgyrBmutations might independently confer resistance (MIC, 16 μg/ml), Ser416 to Ala and Glu466 to Lys. Moxifloxacin-resistant isolates were cross-resistant to ciprofloxacin and levofloxacin, but some moxifloxacin-nonsusceptible isolates remained susceptible to gemifloxacin or nemonoxacin at 0.5 μg/ml. This study found the diversity of toxigenic and nontoxigenic strains ofC. difficilein the health care setting in Taiwan. All isolates tested were susceptible to metronidazole and vancomycin. Fidaxomicin exhibited potentin vitroactivity against all isolates tested, while the more than 10% of Taiwanese isolates with rifaximin MICs of ≥128 μg/ml raises concerns.

scholarly journals Evaluation of the Cepheid Xpert C. difficile/Epiand Meridian Bioscienceillumigene C. difficile Assays for Detecting Clostridium difficile Ribotype 033 Strains

2014 ◽  
Vol 53 (3) ◽  
pp. 973-975 ◽  
Author(s):  
Grace O. Androga ◽  
Alan M. McGovern ◽  
Briony Elliott ◽  
Barbara J. Chang ◽  
Timothy T. Perkins ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Binary Toxin ◽  
Content Type ◽  
Toxin Genes ◽  
Production Animals

Clostridium difficilePCR ribotype 033 (RT033) is found in the gastrointestinal tracts of production animals and, occasionally, humans. TheillumigeneC. difficileassay (Meridian Bioscience, Inc.) failed to detect any of 52C. difficileRT033 isolates, while all strains signaled positive for the binary toxin genes but were reported as negative forC. difficileby the XpertC. difficile/Epiassay (Cepheid).

scholarly journals Nucleic Acid Amplification Test Quantitation as Predictor of Toxin Presence in Clostridium difficile Infection

2017 ◽  
Vol 56 (3) ◽  
Author(s):  
M. J. T. Crobach ◽  
N. Duszenko ◽  
E. M. Terveer ◽  
C. M. Verduin ◽  
E. J. Kuijper
Keyword(s):  
Nucleic Acid ◽  
Clostridium Difficile ◽  
Characteristic Curve ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
Quantitative Results ◽  
Testing Algorithm

ABSTRACT Multistep algorithmic testing in which a sensitive nucleic acid amplification test (NAAT) is followed by a specific toxin A and toxin B enzyme immunoassay (EIA) is among the most accurate methods for Clostridium difficile infection (CDI) diagnosis. The obvious shortcoming of this approach is that multiple tests must be performed to establish a CDI diagnosis, which may delay treatment. Therefore, we sought to determine whether a preliminary diagnosis could be made on the basis of the quantitative results of the first test in algorithmic testing, which provide a measure of organism burden. To do so, we retrospectively analyzed two large collections of samples ( n = 2,669 and n = 1,718) that were submitted to the laboratories of two Dutch hospitals for CDI testing. Both hospitals apply a two-step testing algorithm in which a NAAT is followed by a toxin A/B EIA. Of all samples, 208 and 113 samples, respectively, tested positive by NAAT. Among these NAAT-positive samples, significantly lower mean quantification cycle ( C q ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean C q values of 24.4 versus 30.4 and 26.8 versus 32.2; P < 0.001 for both cohorts). Receiver operating characteristic curve analysis was performed to investigate the ability of C q values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal C q cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, C q values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

scholarly journals Sensitivity of Single-Molecule Array Assays for Detection of Clostridium difficile Toxins in Comparison to Conventional Laboratory Testing Algorithms

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Alice Banz ◽  
Aude Lantz ◽  
Brigitte Riou ◽  
Agnès Foussadier ◽  
Mark Miller ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Single Molecule ◽  
Laboratory Diagnosis ◽  
Case Definition ◽  
Cell Cytotoxicity ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
High Performing ◽  
Molecular Tests

ABSTRACT Guidelines recommend the use of an algorithm for the laboratory diagnosis of Clostridium difficile infection (CDI). Enzyme immunoassays (EIAs) detecting C. difficile toxins cannot be used as standalone tests due to suboptimal sensitivity, and molecular tests suffer from nonspecificity by detecting colonization. Sensitive immunoassays have recently been developed to improve and simplify CDI diagnosis. Assays detecting CD toxins have been developed using single-molecule array (SIMOA) technology. SIMOA performance was assessed relative to a laboratory case definition of CDI defined by positive glutamate dehydrogenase (GDH) screen and cell cytotoxicity neutralizing assay (CCNA). Samples were tested with SIMOA assays and a commercial toxin EIA to compare performance, with discrepancy resolution using a commercial nucleic acid-based test and a second cell cytotoxicity assay. The SIMOA toxin A and toxin B assays showed limits of detection of 0.6 and 2.9 pg/ml, respectively, and intra-assay coefficients of variation of less than 10%. The optimal clinical thresholds for the toxin A and toxin B assays were determined to be 22.1 and 18.8 pg/ml, respectively, with resultant sensitivities of 84.8 and 95.5%. In contrast, a high-performing EIA toxin test had a sensitivity of 71.2%. Thus, the SIMOA assays detected toxins in 24% more samples with laboratory-defined CDI than the high performing toxin EIA (95% [63/66] versus 71% [47/66]). This study shows that SIMOA C. difficile toxin assays have a higher sensitivity than currently available toxin EIA and have the potential to improve CDI diagnosis.

scholarly journals Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool

2015 ◽  
Vol 53 (10) ◽  
pp. 3204-3212 ◽  
Author(s):  
Linan Song ◽  
Mingwei Zhao ◽  
David C. Duffy ◽  
Joshua Hansen ◽  
Kelsey Shields ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Cytotoxicity Assay ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Toxin B ◽  
Content Type ◽  
Toxigenic Culture ◽  
Immunosorbent Assays ◽  
Digital Elisa ◽  
Detection And Quantification

The currently available diagnostics forClostridium difficileinfection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenicC. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinicalC. difficileisolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.

scholarly journals Fidaxomicin and OP-1118 Inhibit Clostridium difficile Toxin A- and B-Mediated Inflammatory Responses via Inhibition of NF-κB Activity

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Hon Wai Koon ◽  
Jiani Wang ◽  
Caroline C. Mussatto ◽  
Christina Ortiz ◽  
Elaine C. Lee ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Intestinal Inflammation ◽  
Inflammatory Responses ◽  
Human Colon ◽  
Toxin A ◽  
Primary Metabolite ◽  
Necrosis Factor Alpha ◽  
Toxin B ◽  
Content Type ◽  
Tnf Α

ABSTRACTClostridium difficilecauses diarrhea and colitis by releasing toxin A and toxin B. In the human colon, both toxins cause intestinal inflammation and stimulate tumor necrosis factor alpha (TNF-α) expression via the activation of NF-κB. It is well established that the macrolide antibiotic fidaxomicin is associated with reduced relapses ofC. difficileinfection. We showed that fidaxomicin and its primary metabolite OP-1118 significantly inhibited toxin A-mediated intestinal inflammation in micein vivoand toxin A-induced cell roundingin vitro. We aim to determine whether fidaxomicin and OP-1118 possess anti-inflammatory effects against toxin A and toxin B in the human colon and examine the mechanism of this response. We used fresh human colonic explants, NCM460 human colonic epithelial cells, and RAW264.7 mouse macrophages to study the mechanism of the activity of fidaxomicin and OP-1118 against toxin A- and B-mediated cytokine expression and apoptosis. Fidaxomicin and OP-1118 dose-dependently inhibited toxin A- and B-induced TNF-α and interleukin-1β (IL-1β) mRNA expression and histological damage in human colonic explants. Fidaxomicin and OP-1118 inhibited toxin A-mediated NF-κB phosphorylation in human and mouse intestinal mucosae. Fidaxomicin and OP-1118 also inhibited toxin A-mediated NF-κB phosphorylation and TNF-α expression in macrophages, which was reversed by the NF-κB activator phorbol myristate acetate (PMA). Fidaxomicin and OP-1118 prevented toxin A- and B-mediated apoptosis in NCM460 cells, which was reversed by the addition of PMA. PMA reversed the cytoprotective effect of fidaxomicin and OP-1118 in toxin-exposed human colonic explants. Fidaxomicin and OP-1118 inhibitC. difficiletoxin A- and B-mediated inflammatory responses, NF-κB phosphorylation, and tissue damage in the human colon.

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